Molecular Detection Assays for Rapid Field-Detection of Rice Sheath Blight.

Ying-Hong Lin,Yi-Jia Lin,Sheng-Chi Chu,Tsai-De Chang,Shih-Mao Shen,Chen-Jie Wen

doi:10.3389/fpls.2020.552916

Abstract

Rhizoctonia solani (Rs), a soil-borne fungal pathogen, can result in rice sheath blight (ShB), which causes yield loss. To prevent outbreaks of ShB and enhance the sustainability of rice production, it is critical to develop a rapid ShB detection method for specific, fast, and on-site disease management. In this study, a reagent for the rapid extraction of this pathogen was developed for on-site detection. The specificity and sensitivity of a novel SMS RS1-F/SMS RS1-R primer set and a ITS1/GMRS-3 reference primer set were tested, while four different extraction protocols for ShB were developed. Moreover, intraday and interday assays were performed to evaluate the reproducibility of the detection methods developed. The results indicated that all of the developed protocols are suitable for use in detecting ShB. In addition, all the samples of infected rice yielded positive Rs detection results when subjected to TaqMan probe-based real-time PCR and SYBR green-based real-time PCR (SMS RS1-F/SMS RS1-R) tests in which automatic magnetic bead-based DNA extraction was performed. These results indicated that the two molecular detection protocols were suitable for the field diagnosis of ShB for all asymptomatic and symptomatic rice samples.

Highlights

Paddy rice (Oryza sativa), which is one of the world’s main food crops, takes up nearly 11% of the arable land worldwide, in addition to being the food that the largest proportion of people eat as their primary food (FAOSTAT, 2019)
Rhizoctonia solani (Rs). solani can be amplified and visualized by the bands at 118 and 550 bp when using the primer pairs internal transcribed spacers 1 (ITS1)/GMRS-3 and SMS RS1-F/SMS RS1-R (Figure 1), whereas, other pathogens, such as P. oryzae, B. oryzae, F. verticillioides, C. gloeosporioides, C. lagenarium, F. oxysporum f. sp. cubense, F. oxysporum f. sp. niveum, F. acuminatum, F. oxysporum f. sp. gladioli, and A. alternata, do not elicit the same results as R. solani. These R. solani isolates were identified as R. solani AG1-IA based on the phylogenetic analysis of their internal transcribed spacer (ITS) sequence (Figure 2). These results prove that the primer we designed, SMS RS-1F/SMS RS1-R, and the reference primer ITS1/GMRS-3 (Johanson et al, 1998) have specificity for the pathogen of sheath blight (ShB)
The PCR sensitivity levels of using SMS RS-1F/SMS RS1-R (Figures 3A,C,E) and ITS1/GMRS-3 (Figures 3B,D,F) to detect mycelial genomic DNA (gDNA), standard DNA, and sclerotial gDNA were 10−5 ng, 102 copies, and 10−4 ng, respectively. These results indicate that the sensitivity levels of the primers we designed, SMS RS1F/SMS RS1-R, and that of the reference primer, ITS1/GMRS3, are similar

Summary

Introduction

Paddy rice (Oryza sativa), which is one of the world’s main food crops, takes up nearly 11% of the arable land worldwide, in addition to being the food that the largest proportion of people eat as their primary food (FAOSTAT, 2019). The counterpart was already known and reported in the 20th century, and the yield of paddy rice dropped a lot in the United States, Japan, and China (Ou, 1985; Groth, 2005; Prasad and Eizenga, 2008; Bernardes-de-Assis et al, 2009; Zhang et al, 2009; Chen et al, 2012). It has become the primary pathogen affecting rice production for (Yuan et al, 2018). To develop a rapid method for detecting ShB, is vital and essential

Methods

Results

Discussion

Conclusion